Connectors for pneumatic devices in microfluidic systems

ABSTRACT

A vacuum line may be placed within the vacuum port, and configured to deliver negative pressure to the vacuum port. A vacuum holding area is created in the volume between the outer seal and each of the sealing members when the inner surface of the body is placed against a substrate. When the vacuum line is activated, a vacuum is created within the vacuum holding area, creating a positive seal between the body and the substrate.

This application is a divisional of U.S. patent application Ser. No.15/006,765 filed Jan. 26, 2016, which claims priority of U.S.Provisional Application Ser. No. 62/130,089 filed Mar. 9, 2015, thedisclosures of which are incorporation herein by reference.

FIELD OF THE DISCLOSURE

The disclosure generally relates to microfluidic devices and methods forcell culture. In particular, the disclosure relates to connectors usedin the pneumatic control of microfluidic devices and cell culture.

BACKGROUND

The ability to grow and maintain cells in vitro was a significantmilestone in the biological sciences. However, traditional cell culturetechniques lack the ability to analyze single cells, as opposed to bulkcultures. Population-averaged bulk assays are often inaccurate ormisleading due to natural cell-to-cell variability. Further, cellsignaling and other biochemical parameters constantly change, makingdynamic analysis of cells crucial in understanding how a biologicalsystem operates. In response to these limitations, microfluidic cellculture systems have been developed that allow for high throughput andmultiplexed culture and analysis of individual cells.

Microfluidic cell culture is a promising technology for applications indrug screening, tissue culturing, toxicity screening, and biologicresearch and can provide improved biological function, higher qualitycell-based data, reduced reagent consumption, and lower cost. The mostcommon approach for manufacturing microfluidic devices is softlithography of polydimethylsiloxane (PDMS), which allows structures ofmicrometer resolution to be molded from a hard master. PDMS-basedculture systems and devices may include a variety of structures,including various kinds of channels, chambers, barriers, and valves.Each of these components may be networked together in variousconfigurations to create a “lab on a chip” device that can be utilizedto conduct a variety of biological experiments. Further, microfluidiccell culture systems can be highly multiplexed, allowing for multipleconditions or samples to be tested on a single device.

Key benefits of microfluidic cell culture include improved biologicalfunction, higher-quality cell-based data, reduced reagent consumption,and lower cost. Further, high quality molecular and cellular samplepreparations are important for various clinical, research, and otherapplications. In vitro samples closely representing their in vivocharacteristics can potentially benefit a wide range of molecular andcellular applications. Handling, characterization, culturing, andvisualization of cells or other biologically or chemically activematerials (such as beads coated with various biological molecules) havebecome increasingly valued in the fields of drug discovery, diseasediagnoses and analysis, and a variety of other therapeutic andexperimental work.

The relatively small scale and multiplexed nature of microfluidicdevices results in high applicability to automation. Automated systemsare particularly useful in the pharmaceutical industry, which relies onhigh throughput screening of libraries of chemical compounds to findpotential drug candidates. By using microfluidic devices, highthroughput screening can test many discrete compounds in parallel sothat large numbers of test compounds are screened for biologicalactivity simultaneously. In such systems, pneumatic control is oftenused to load cells and drive other actions on a microfluidic device.However, imperfect sealing of a pneumatic control system to amicrofluidic device may result in improper pressures being applied tothe device, thus biasing the results of the analysis. Connectionsbetween the pneumatic control system and microfluidic device, such asgas line tubing, may also become contaminated, requiring eitherdisposal, or extensive and manual cleaning.

SUMMARY

The problems of the prior art are addressed by a novel design of apneumatic connector for interfacing a microfluidic control and analysissystem with a microfluidic device. Embodiments of pneumatic connectorsaccording to the disclosure may be in in communication with either endof a tubing, such as 10-line ribbon tubing, used to supply gases,fluids, or other media from a pneumatic control system to a microfluidicdevice. Pneumatic connectors may be removable and secured using anexisting in-line vacuum force provided via the tubing and pneumaticcontrol system. In certain embodiments, the pneumatic connectors may beremovable and secured using magnetic force. In still furtherembodiments, the pneumatic connectors may use mechanical attachmentmeans, such as thumb screws and the like. Pneumatic connectors maysimultaneously establish multiple secure connections from a pneumaticcontrol system to a microfluidic device. The connections may beconfigured to deliver variable pressure to control fluidic flow on themicrofluidic device. At least one connection may be configured todeliver negative pressure to create a vacuum. In certain embodiments,pneumatic connectors may be configured to engage with a rigid pneumaticmanifold that interfaces with a consumable microfluidic plate designedfor live cell culture and imaging. Accordingly, in these embodiments,the vacuum may be used to seal the pneumatic manifold to themicrofluidic plate, and also to seal the pneumatic connector to thepneumatic manifold. In still further embodiments, pneumatic connectorsmay be configured to engage with a pneumatic interface of the pneumaticcontrol system. Further, in certain embodiments, the connector maycomprise filters for preventing the backflow of liquids into acontroller. Accordingly, the novel design results in a removable,repeatable, and reliable pneumatic connector located directly at aconvenient interface between the pneumatic controller and themicrofluidic plate. When used in an automated system, embodiments ofpneumatic connectors according to the disclosure greatly ease userworkflow and substantially reduce the possibility of malfunctions.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the present disclosure, reference is madeto the accompanying drawings, which are incorporated herein by referenceand in which:

FIG. 1 is a system diagram of an embodiment of a microfluidic controland analysis system according to the disclosure;

FIG. 2 is a top view of an embodiment of a microfluidic plate within themicrofluidic control and analysis system of FIG. 1;

FIG. 3 is a diagram depicting an embodiment of a cell culture areawithin the microfluidic plate of FIG. 2;

FIGS. 4A-B are perspective views of an embodiment of a pneumaticmanifold according to the disclosure as it is positioned onto themicrofluidic plate of FIG. 2;

FIG. 5 is a top view of the pneumatic manifold of FIGS. 4A-B;

FIG. 6 is a perspective view of an embodiment of a pneumatic connectoraccording to the disclosure;

FIG. 7 is a perspective view of the pneumatic connector of FIG. 6 andthe pneumatic manifold of FIG. 5;

FIG. 8 is a front view of the pneumatic connector of FIG. 6;

FIGS. 9A-B are cross-sectional views of the pneumatic connector of FIG.6 and the pneumatic manifold of FIG. 7 as the pneumatic connector isplaced in communication with the pneumatic manifold;

FIG. 10 is a top view of an embodiment of a pneumatic interface forreceiving a pneumatic connector located on an embodiment of a pneumaticmanifold according to the disclosure;

FIGS. 11A-B are perspective views of another embodiment of a pneumaticinterface located on a pneumatic control system for receiving thepneumatic connector of FIG. 6;

FIGS. 12A-B are perspective views of another embodiment of a pneumaticconnector according to the disclosure in the disengaged and engagedstates, respectively;

FIGS. 13A-B are front views of the pneumatic connector of FIGS. 12A-B inthe disengaged and engaged states, respectively;

FIG. 14 is an exploded front view of the removable pneumatic connectorof FIGS. 12A-B;

FIG. 15 is a perspective view of an embodiment of a cleaning plateaccording to an embodiment of the disclosure;

FIG. 16 is a top view of the cleaning plate of FIG. 15; and

FIG. 17 is a top view of a pneumatic manifold positioned over thecleaning plate of FIG. 15.

DETAILED DESCRIPTION

The detailed description set forth below in connection with the appendeddrawings is intended as a description of embodiments and does notrepresent the only forms which may be constructed and/or utilized.However, it is to be understood that the same or equivalent functionsand sequences may be accomplished by different embodiments that are alsointended to be encompassed within the spirit and scope of thedisclosure, such as removable pneumatic connectors and systems usingdifferent geometries, materials, number of connections, and otheralignment or mounting features in order to facilitate mounting,automation, or simple operator use.

Microfluidic Plate Control and Analysis System

Microfluidic cell culture systems provide a powerful tool to conductbiological experiments. FIG. 1 illustrates an embodiment of amicrofluidic plate control and analysis system 10 according to thepresent disclosure. The microfluidic plate control and analysis system10 comprises a microfluidic plate 100 positioned on the stage of aninverted microscope 20. Cell culture or other processes occurring on theplate 100 may be observed using the inverted microscope 20. The plate100 is in communication with a pneumatic controller 40 via tubing 30,which may comprise 10-line ribbon tubing. The tubing 30 may alsocomprise other forms of communication and connections between thepneumatic controller 40 and microfluidic plate 100, such as individualgas line tubing, electrical wiring, heating elements, networkingcomponents, and the like. The pneumatic controller 40 may be configuredto interact with the microfluidic plate 100 by using the tubing 30 tosupply a gas or liquid to the plate 100, control the temperature of theplate 100, or perform other desired functions. The controller 40 isfurther in communication with a computer 60 via a network connection 50.The computer 60 may be configured to display and/or analyze image datafrom the microscope 20, record actions taken by the pneumatic controller40, and instruct the pneumatic controller 40 to take actions accordingto a protocol.

In this embodiment, the microfluidic plate 100 comprises a glass bottomfor imaging, and may be configured to fit within the stage holder of theinverted microscope 20. In certain embodiments, the microfluidic plate100 has dimensions corresponding to a Society for Biomolecular Screening(SBS) standard 96-well plate. The microfluidic plate 100 may use anapplication-specific design depending on the type of experiment desired,such as cell culture, solution exchange, or comparison of conditions. Incertain embodiments, the microfluidic plate 100 may be a CellASIC® ONIXMicrofluidic Plate for Live Cell Analysis, commercially available fromEMD Millipore Corporation. Further, the microfluidic plate 100 may bemultiplexed, allowing for a single microfluidic plate 100 to performseveral individual or related experiments, either simultaneously orsequentially.

The tubing 30 may be configured or utilized for a particular purpose,such as supplying a gas or liquid to the microfluidic plate 100. In thisembodiment, the tubing 30 comprises 10-line ribbon tubing configuredsuch that eight of the lines provide variable pressure to themicrofluidic plate 100, one line provides a desired gas environment, andone line provides negative pressure to create a vacuum. In certainembodiments, the tubing 30 may further comprise a connection (e.g., anelectrical connection) in communication with a heating element or heatexchanger in communication with the microfluidic plate 100, thusincubating the microfluidic plate 100 to a desired temperature. Whilethe embodiments described in this disclosure utilize a 10-line ribbontubing, various other forms of connections and communication between thepneumatic controller 40 and microfluidic plate 100 may be used,including those of greater or fewer lines, or utilizing other means ofdelivering pressure, gas, vacuum, and/or heat.

Each line of the 10-line ribbon tubing may be in communication with thecontroller 40, which may comprise a plurality of ports configured togenerate pressure or vacuum, regulate pressure, open or close valves,and/or supply a gas environment (e.g., 5% CO₂) having a desiredtemperature and humidity. The controller 40 may further comprise aheating controller that instructs a corresponding heating element incommunication with the microfluidic plate 100 to raise or lower thetemperature of the microfluidic plate 100. For example, the heatingcontroller may be configured to maintain the temperature of themicrofluidic plate at 37° C., mimicking in vivo conditions. In thisembodiment, the controller 40 is a CellASIC® ONIX Microfluidic ControlSystem (commercially available from EMD Millipore Corporation), which isable to supply positive pressure up to 10 PSI and negative pressure of−8.2 PSI. However, any suitable controller that is able to provide anyof variable pressure, a desired gas environment, or temperature controlfor a microfluidic device may be used.

The computer 60 is in communication with the controller 40 over anetwork connection 50. In this embodiment, the network connection 50comprises a USB connection. However, the network connection 50 may beany form of connection enabling communication between the controller 40and computer 60, including serial, parallel, and Ethernet connections.Further, in certain embodiments, the controller 40 and computer 60 maycomprise a single unit. In this embodiment, the network connection 50may be an integral component.

In this embodiment, the computer 60 includes software configured tomanage various aspects of the microfluidic plate control and analysissystem 10. The computer 60 may be configured to operate the controller40 according to a protocol for an experiment. For example, the computer60 can send control signals to the controller 40 instructing thecontroller 40 to provide variable pressure to the microfluidic plate100, or take other actions, according to a either a pre-determined ordynamic schedule. Further, the computer 60 may be configured to receiveuser input and modify protocols, including the ability to set flowsequences, set desired pressures, or store programs and protocols. Thecomputer 60 may also be used to determine the overall system status.However, in certain embodiments, these features may also be implementedwholly or partly within the controller 40.

In certain embodiments, the computer 60 may further be in communicationwith a digital camera attached to the inverted microscope 20. In theseembodiments, the computer 60 may include the capability to display,monitor, and track images captured by the digital camera of themicrofluidic plate 100. This feature is particularly useful forlong-term live cell analyses, wherein processes may take days andinteresting events may occur during off hours. Further, in largerautomated systems, this feature can be used to track conditions atdesignated time points for a plurality of samples without a need forhuman intervention.

Microfluidic Culture Plate

FIG. 2 illustrates the microfluidic plate 100 within the microfluidiccontrol and analysis system 10 of FIG. 1 in further detail. In thisembodiment, the microfluidic plate 100 may be PDMS-based and maycomprise a plurality of independent assay units (i.e., the 4 rowslabeled “A”-“D”). Each assay unit may comprise a plurality of fluidicchannels 102 in communication with a culture chamber 104. Cells or otherfluids may be loaded into the culture chamber 104 via a cell inlet well106 (e.g., the well labeled “A6”). Further, various solutions orreagents may be provided to the culture chamber 104 via a plurality ofsolution inlet wells 108 (“A2”-“A5”) and a gravity perfusion well 110(“A1”). A viewing window 112 is formed over the culture chamber 104,allowing for placement of the lens of a microscope (e.g., the invertedmicroscope 20 of FIG. 1) to view the cells or other processes takingplace within. Each assay unit may further comprise a waste outlet well114 (“A7”) for waste from the culture chamber 104, and a perfusionoutlet well 116 (“A8”) for waste from the solution inlet wells 108 andgravity perfusion well 110.

A plurality of sidewalls 105 extending upward from the plate 100 areformed around the wells, culture chamber 104, and viewing window 112,isolating these features from one another. At least some of thesidewalls 105 extend to the top of the plate 100, such that placing amanifold over the plate 100 results in the sidewalls 105 being incontact with the manifold. As will be described further below, thisfeature may be used to deliver isolated pneumatic pressure to each wellvia a pneumatic manifold, provide a desired gas environment to theculture chambers 104, or create a vacuum within other areas of the plate100.

FIG. 3 illustrates the culture chamber 104 in further detail. The cellinlet well 106 is in direct communication with the culture chamber 104,allowing for free flow of cells from the cell inlet well 106 into theculture chamber 104. In contrast to the cell inlet well 106, the fluidicchannels 102 connecting the solution inlet wells 108 and gravityperfusion well 110 are separated from the culture chamber 104 by aperfusion barrier 118. In this embodiment, the perfusion barrier 118 isa combination of solid structures and passages smaller than the fluidicchannels 102 that separates the fluidic channels 102 from the culturechamber 104. The perfusion barrier 118 is designed to keep cells, otherculture items, and gels from migrating into the fluidic channels 102,while allowing some fluidic flow through diffusion, perfusion, or anycombination of mass transfer mechanisms that is generally of a muchhigher fluidic resistance than the fluid flow in the flow channels. Inthis way, media and reagents can be supplied to the culture chamber 104without the risk of blocking the fluidic channels 102.

The microfluidic plate 100 is prepared for use by first priming thefluidic channels 102 with a desired buffer, such as sterile PBS. Next,10 μL of a desired cell suspension is pipetted into the cell inlet well106. Aspirating the waste outlet well 114 causes the cell suspension toload into the culture chambers 104 through capillary action. Once in theculture chamber 104, cells may be perfused with media supplied to thegravity perfusion well 110, or exposed to reagents or other chemicalssupplied to any of the solution inlet wells 108. As the plate 100includes four independent assay units, up to four different samples ofcells may be independently cultured on a single plate 100. The status ofcell culture and response may be observed, for example, by viewing eachculture chamber 104 through the viewing window 112 with a microscope.

Once cells are sufficiently cultured, a variety of experiments may beconducted using the microfluidic plate 100. For example, the solutioninlet wells 108 can be used for solution switching experiments, whereincells are sequentially exposed to various solutions and the resultingcellular response is analyzed. To expose cells within the culturechamber 104 to a desired solution, an amount of that solution (e.g., 10μL) is pipetted into a solution inlet well 108 (e.g., A2). The solutionthen traverses the fluidic channels 102 and perfuses through theperfusion barrier 118 and into the culture chamber 104. Subsequently,the cells may be exposed to other solutions via the other solution inletwells and similarly observed. In addition to solution switching, thesolution inlets may also be used for automated staining and washingprotocols, and on-demand fixation by flowing fixative into the culturechamber 104 during imaging.

Further, it should be noted that while the present disclosure refers topneumatic control of the microfluidic plate 100, embodiments of thedisclosure may be used for any form of microfluidic device, plate, orcontrol and analysis system. Various embodiments are considered to bewithin the scope of the disclosure.

Pneumatic Manifold

Simple gravity-driven perfusion may be used to both culture cells andexpose cells to various reagents. While gravity-driven perfusion allowsfor an operator to conduct an experiment using only a microfluidic plate100 without any additional hardware (e.g., the controller 40 and/orcomputer 60), it lacks a degree of fine control and also requirescontinuous monitoring by an operator. Accordingly, pneumatic control byway of a pneumatic manifold 120, as in the embodiment shown in FIGS.4A-B, may also be used to control loading of cells and reagents on themicrofluidic plate 100. The pneumatic manifold 120 may be mated to themicrofluidic plate 100 in order to finely control cell loading,perfusion of media, and solution exposure by providing variable pressureto each of the wells of the microfluidic plate 100.

FIGS. 4A-B illustrate the placement and sealing of the pneumaticmanifold 120 to the microfluidic plate 100. The manifold 120 maycomprise a cyclic olefin copolymer body configured to be positioned overthe microfluidic plate 100 and further comprises a plurality of channels122 which may be used to supply a gas or liquid or deliver negativepressure to the microfluidic plate 100. Each of the channels 122 is incommunication with the tubing 30, which as described above may comprise10-line tubing in communication with a suitable pneumatic controller(such as the pneumatic controller 40 of FIG. 1). The manifold 120further comprises a soft gasket 132, which should first be cleaned,e.g., with 70% ethanol, and then blotted dry. The plate 100 is thenpositioned on a flat surface, and the manifold 120 is aligned and setover the wells, as shown in the embodiment of FIG. 4B. Once in place,negative pressure is supplied to at least one of the channels 122 (viaone of the gas lines of the tubing 30) such that a vacuum is createdbetween the microfluidic plate 100 and pneumatic manifold 120. As thevacuum is created, an operator (or automated instrument) may press themanifold 120 against the plate 100 for several seconds to ensure uniformcontact of the gasket 132. A proper seal forms as the volume between thewells, sidewalls 105, plate 100, and manifold 120 becomes a vacuum. Oncea proper seal has been formed, the vacuum should be maintained by anappropriate negative pressure (e.g., −8.2 PSI) to maintain a positiveseal and vacuum throughout the course of the experiment. In certainembodiments, the pneumatic manifold 120 may be an F84 Manifold forCelIASIC® ONIX, commercially available from EMD Millipore Corporation.

FIG. 5 illustrates the pneumatic manifold 120 and plurality of channels122 in further detail. In the embodiment shown, each channel 122includes a channel inlet 124 on a top side of the manifold 120, which isin communication with a respective gas line, e.g., a line from the10-line ribbon tubing comprising the tubing 30 of FIG. 1. Each channel122 is further in communication with at least one channel outlet 126located on a bottom side of the pneumatic manifold 120. Each channeloutlet 126 is positioned such that when the pneumatic manifold 120 ispositioned over the microfluidic plate 100, each channel outlet 126 ispositioned over a particular well (e.g., the wells of the microfluidicplate 100 of FIG. 2). Further, the sidewalls 105 of the microfluidicplate 100 are in contact with the underside of the manifold 120, thusisolating each channel outlet 126 such that it is only communicationwith a single well or area of the microfluidic plate 100.

In this embodiment, the pneumatic manifold 120 is configured withsufficient channels 122 and channel outlets 126 to match the number ofwells and assay units on the plate 100. Eight of the channels 122 (i.e.,the channels 122 labeled “V1”-“V8”) include four channel outlets 126,corresponding to the four independent assay units of the microfluidicplate 100 of FIG. 2. Thus, a single channel inlet 124 can be used toapply pressure to a particular well of four assay units on themicrofluidic plate 100, controlling flow rates through the fluidicchannels 102 of the microfluidic plate 100. However, in certainembodiments, the number and location of channels 122, channel inlets124, and channel outlets 126 may be varied to match the configuration ofa particular microfluidic plate or control system or other needs for anexperiment.

The plurality of channels 122 may further comprise a gas environmentchannel 128, which includes a channel outlet 126 positioned over theviewing window 112 and culture chambers 104 (as shown in FIG. 2). Thegas environment channel 128 can be used to provide atmospheric controlfor the microfluidic plate 100 and bathe cells in the culture chamber104 with a specified gas environment. As previously described, themicrofluidic plate 100 comprises a gas-permeable device layer (i.e.,PDMS) over a glass bottom. Thus, gases provided to the microfluidicplate 100 can be delivered to the culture chambers 104 through thegas-permeable device layer by diffusion. In certain embodiments, the gasdelivered via the gas environment channel 128 comprises 5% CO₂; however,any gaseous mixture, such as mixtures including oxygen and/or nitrogen,may be used. By continuously flowing gas through the gas environmentchannel 128, a stable gas environment for culturing cells within theculture chambers 104 is maintained. Thus, the gas environment channel128 provides a means for controlling the environment within the culturechambers 104 other than placing the microfluidic plate 100 into anincubator. This results in the manifold 120 becoming a“micro-incubator,” independent of the ambient air, allowing continuousmedium perfusion and preventing evaporation.

The plurality of channels 122 may further comprise a vacuum channel 130.The channel outlet 126 for the vacuum channel 130 is positioned in anarea between the wells and sidewalls 105 of the microfluidic plate 100.Thus, supplying negative pressure to the vacuum channel 130 when themanifold 120 is positioned over the microfluidic plate 100 creates avacuum in the volume between the wells, sidewalls 105, manifold, and themicrofluidic plate 100, thus sealing the manifold 120 to the plate 100.

Thus, by using the pneumatic manifold 120, pressure can be applied toindividual wells to drive cell loading, solution switching, or perfusionof media. Cells may be incubated with a suitable gas environment, and avacuum ensures that the manifold 120 remains sealed to the microfluidicplate 100. Further, connecting the channel inlets 124 to a controllerand corresponding computer (such as the controller 40 and computer 60 ofFIG. 1) can be used to automate various protocols and experimentsrunning on the microfluidic plate 100.

As noted above, in this embodiment, the tubing 30 comprises a gas lineribbon tubing having ten lines: eight for pressure control, one foratmosphere, and one for vacuum. However, various numbers and types ofconnections may be utilized according to embodiments of the disclosure.For example, in certain embodiments, the tubing 30 may provide a liquidto a microfluidic plate 100 or other device. In certain embodiments, thetubing 30 may provide both liquid and pressure control lines, or providetemperature control for the microfluidic plate 100.

Connections Between the Manifold and the Controller

As noted above, the tubing 30 connecting the manifold 120 and thecontroller 40 may comprise a plurality of gas lines, such as 10-lineribbon tubing. In certain embodiments, the tubing 30 may be permanentlyconnected to both the pneumatic controller 40 and the manifold 120. Thetubing 30 may also be removable from either the pneumatic controller 40or manifold 120, or both, by a variety of mechanisms, including bypneumatic, magnetic, mechanical attachment, and the like.

A. First Embodiment of a Removable Pneumatic Connector

FIG. 6 illustrates an embodiment of a pneumatic connector 150. Thepneumatic connector 150 may be used as an attachment mechanism forremovably securing the tubing 30 to a pneumatic manifold, such as thepneumatic manifold 120 of FIG. 5. While in this embodiment, thepneumatic connector 150 is positioned between the tubing 30 and manifold120, in certain embodiments the pneumatic connector 150 may bepositioned between the tubing 30 and the controller 40. In still furtherembodiments, pneumatic connectors 150 may be situated at both positions.

In this embodiment, the pneumatic connector 150 uses an existing vacuumline 32 on the tubing 30 to removably secure the pneumatic connector 150to the manifold 120. However, in certain embodiments, the pneumaticconnector 150 may use alternate lines separate from the tubing 30 fordelivering vacuum or negative pressure to removably secure the connector150.

The pneumatic connector 150 may comprise a body 152 in communicationwith tubing 30, such as the 10-line ribbon tubing in communication witha pneumatic controller (such as the controller 40 of FIG. 1). In theembodiment shown, the body 152 comprises clear PDMS molded onto apolycarbonate sheet. The pneumatic connector 150 may be configured to bepositioned onto the surface of a pneumatic manifold 120 having acorresponding interface as a substrate, such that each gas line of thetubing 30 is in communication with a respective channel inlet 124 on themanifold 120 (as shown in the embodiment of FIG. 7). In this embodiment,the body 152 comprises a rounded rectangular shape. However, a varietyof other shapes may be used to accommodate various configurations oftubing 30 and a corresponding interface and substrate according tovarious embodiments of the disclosure.

FIG. 8 illustrates various aspects of the pneumatic connector 150 infurther detail. The body 152 of the pneumatic connector 150 comprises aninner surface 164 that is configured to be placed onto a correspondingsubstrate, such as the channel inlets 124 on the pneumatic manifold 120of FIG. 5. The body 152 of the pneumatic connector 150 further comprisesa plurality of bores 156 passing through the body 152 such that thebores 156 are exposed to the inner surface 164. Some of the bores 156may be surrounded by sealing members, such as seals 158. The innersurface 164 may further comprise an outer seal 160 which surrounds eachof the bores 156. In this embodiment, at least one of the bores 156 isutilized as a vacuum bore or vacuum port 162, which lacks acorresponding seal 158.

Each of the bores 156 is in communication with a corresponding gas linefrom the tubing 30. As previously described, the tubing 30 in thisembodiment comprises ten gas lines: pressure controlled lines 1-8, a gasenvironment line, and a vacuum line. Each gas line is placed within acorresponding bore 156. Whereas each bore 156 for the pressurecontrolled lines and gas environment line includes a corresponding seal158, the vacuum line placed within the vacuum port 162 does not have aseal.

Placing the connector 150 against a corresponding interface or surfaceon the pneumatic manifold 120 causes the seals 158 and outer seal 160 tocome into contact with that substrate. When placed against such asubstrate, a vacuum holding area 154 is formed. The vacuum holding area154 comprises a space or volume having edges defined by the outer seal160, inner seals 158, the inner surface 164, and the correspondingsubstrate against which the connector 150 is placed. Further, the innerseals 158 create a fluid tight separation between each gas line withineach bore 156. However, because the vacuum port 162 does not include aseal 158, the vacuum port 162 is in fluid communication with the vacuumholding area 154. Thus, supplying negative pressure to the vacuum port162 (e.g., via tubing 30 in communication with the controller 40 ofFIG. 1) creates a vacuum within the vacuum holding area 154.Accordingly, the difference in pressure between the outside environmentand the vacuum holding area 154 seals the pneumatic connector 150 to thesurface, creating a secure connection that can be actuated bydeactivating and activating the vacuum line in communication with thevacuum port 162.

FIGS. 9A-B illustrate the pneumatic connector 150 as it is placedagainst a manifold 120. In use, the pneumatic connector 150 is placedagainst the channel inlets 124 of the pneumatic manifold 120. Each ofthe bores 156 are configured to be positioned over a correspondingchannel inlet 124. Actuating the pneumatic connector 150 by creating avacuum within the vacuum holding area 154 thus places each gas line ofthe tubing 30 in communication with a respective channel inlet 124. Theseals 158 substantially prevent communication between each channel inlet124, minimizing any cross-talk or leakage between channels. Further, inthis embodiment, the pneumatic manifold 120 also includes the vacuumchannel 130. Thus, creating a vacuum in the vacuum holding area 154further supplies a vacuum to the vacuum channel 130 via itscorresponding channel inlet 124, sealing the manifold 120 to amicrofluidic plate. In this way, the pneumatic connector 150 is able toutilize an existing vacuum line to both hold the pneumatic connector 150in place against the pneumatic manifold 120, and simultaneously securethe pneumatic manifold 120 to the microfluidic plate. Once secured, theconnector 150 may then be used to deliver variable pressure, gas,liquid, or a specified gas environment to various components of themicrofluidic plate.

In this embodiment, the seals 158 may comprise O-rings, and the outerseal 160 may comprise a gasket, each of which have a similar thickness,height, and compressibility ratio. However, in certain embodiments,other kinds of seals may be used, provided that the seals sufficientlyprevent fluid communication between the bores 156 and thus prevent anyleakage between the gas lines of the tubing 30. Further, other kinds ofseals may be used provided that a suitable vacuum holding area 154 iscreated that can sustain vacuum to secure the connector 150 to themanifold 120. Ideally, the choice of seals 158 should result in low leakrates, such as less than 0.1 mL/minute when the gas lines are delivering10 PSI and the vacuum line is held at −8.2 PSI. While in thisembodiment, the tubing 30 comprises ten lines including one vacuum line,in other embodiments, various numbers and combinations of lines may beused, provided that the combination results in a secure connection tothe manifold 120.

As described above, in use, the pneumatic connector 150 is placedagainst the pneumatic interface 134 and the vacuum is activated.Alternately, the vacuum may be activated prior to placing the pneumaticconnector 150 against the pneumatic interface 134. The active vacuumline readily grips, holds, and compresses the seals 158 and the outerseal 160 against the substrate of the manifold 120, drawing theconnector 150 towards the substrate of the manifold, creating a fluidtight seal and establishing a confident connection of all pressurelines, substantially reducing any leakage or “cross-talk.” Due to thevacuum holding area 154, seals 158, and outer seal 160, the pneumaticconnector 150 allows for variable connector alignment and consistentsealing, independent of the skill of the operator. Further, misalignmentcan be detected by a drop in pressure or inability to provide pressureor gas to any of the corresponding channel inlets 124. This detectionmay be made by a controller or computer in communication with the tubing30, such as the controller 40 or computer 60, respectively, of FIG. 1.

In certain embodiments, the manifold 120 may comprise additionalfeatures to aid proper alignment of the connector 150 to the manifold120 and channel inlets 124. FIG. 10 illustrates an embodiment of apneumatic manifold 120 for use with the pneumatic connector 150 thatincludes a pneumatic interface 134. In this embodiment, the pneumaticinterface 134 comprises a tab extending from the pneumatic manifold 120including the channel inlets 124 in communication with the channels 122,gas environment channel 128, and vacuum channel 130 of the pneumaticmanifold 120. The pneumatic interface 134 may also comprise an alignmentmechanism 136 for proper alignment, such as a notch or ridge shaped toreceive the pneumatic connector 150 and to hold it in place when it ispositioned over the pneumatic interface 134. However, in certainembodiments, the pneumatic interface 134 may simply comprise theplurality of channel inlets 124 without any additional features (e.g.,the embodiment shown in FIG. 5).

FIGS. 11A-B illustrate another embodiment of a pneumatic interface 170for use with a pneumatic connector 150. The pneumatic interface 170 maybe located either between the manifold and tubing, or between thecontroller and tubing. In this embodiment, the pneumatic interface 170is connected to a pneumatic controller, such as the controller 40 ofFIG. 1. As shown, the pneumatic interface 170 comprises a body 172having a mating surface 174 comprising a plurality of channel inlets 176in communication with channels within the controller 40. The body 172and mating surface 174 further comprise a rounded rectangle shape so asto accommodate placement of the pneumatic connector 150. In use, thepneumatic connector 150 is placed over the interface 170 such that thevacuum line 32 of the tubing 30 is in communication with a respectivevacuum channel of the interface 170. The vacuum line 32 is thenactivated, securing the connector 150 to the interface 170 in the manneras described above.

In certain embodiments, the microfluidic plate 100 or pneumatic manifold120 may not include a vacuum channel 130. Thus, in these embodiments, apneumatic connector 150 according to the disclosure may comprise avacuum port 162 that is not in communication with a vacuum channel 130of the manifold 120. In these embodiments, the vacuum port 162 servesonly to secure the connector 150 to the manifold, thus placing each bore156 in communication with a respective channel inlet 124.

The pneumatic connector 150 results in a variety of advantages. Forexample, the pneumatic connector 150 allows for the manifold 120 to beeasily cleaned, or even to be used as a plate lid or cover during sampletransfer within labs. Because the pneumatic connector 150 utilizes theexisting vacuum line to hold itself in place during use, no additionalor pneumatics are required. Thus, the pneumatic connector 150 canutilize pre-existing hardware that can also be used to control amanifold having an umbilical-style, or permanent, connection. Further,by establishing a near-automatic holding force, the pneumatic connector150 eases operator workflow and reduces the chances of malfunction.

The pneumatic connector 150 is particularly advantageous in microfluidiccontrol system environments utilizing automation. As noted above, inthis embodiment, the microfluidic plate 100 comprises an SBS-compliant96 well format, and thus various “off-the-shelf” machines can be used tocreate an automated system. In one embodiment, an automated systemincludes a robotic arm or plate handler that moves the microfluidicplate 100 to a particular station. The microfluidic plate 100 may bealready prepared and include the pneumatic manifold 120; however incertain embodiments, the automated system may dispense liquids into thewells of the plate 100 and also introduce the pneumatic manifold 120.The pneumatic connector 150 would then be mechanically introduced by tothe pneumatic interface 134. Activating the vacuum line thenautomatically secures the pneumatic connector 150 to the pneumaticinterface 134, establishing a secure, vacuum-held connection without anyexternal or manual intervention. This feature has a significantadvantage over connectors that use mechanical attachment or clampingmeans. Further, the pneumatic connector 150 presents a reliable andrepeatable connector directly at a point of connection of the pneumaticmanifold 120.

As noted above, the vacuum holding area 154 and seals 158 physicallyseparate each gas line. However, pressure leakage may still occur due toa misaligned, broken, or otherwise incomplete seal. If unnoticed, thispressure leakage may lead to incorrect pressures being applied to eachchannel 122, potentially biasing the results of an experiment beingperformed on the microfluidic plate 100. One advantage of using aremovable pneumatic connector 150 is that any incomplete seals resultingin pressure leakage between gas lines can be recognized as an aberrationin vacuum pressure within the vacuum holding area 154. In certainembodiments, the controller 40 and/or computer 60 are configured torecognize deviations in pressure within the vacuum holding area 154 andreport this information, e.g., via an alert or other means, to anoperator. Thus, the operator may then take corrective action, such asreseating the pneumatic connector 150, to ensure a positive seal.

B. Second Embodiment of a Removable Pneumatic Connector

As noted above, the tubing 30 of FIG. 1 may also be removable from thecontroller 40 by a variety of means. For example, at the interfacebetween the tubing 30 and controller 40, a variety of attachment meansmay be used, such as pneumatic, magnetic, mechanical attachment, and thelike. FIGS. 12A-14 illustrate another embodiment of a removablepneumatic connector 200 according to the disclosure. The pneumaticconnector 200 may be positioned between the tubing 30 and the controller40, and may be configured to removably secure the pneumatic connector200 to the controller 40. Further, in this embodiment, the pneumaticconnector 200 further comprises in-line filters, which may be used toallow the passage of gas, but prevent fluid flow.

As shown in the embodiment of FIGS. 12A-B, the connector 200 maycomprise a housing 202. The housing 202 may comprise clear PDMS, moldedplastic, or another appropriate material. The housing 202 furthercomprises a tubing end 204 and an engagement end 206. A plurality ofmale ports 230 are disposed within and pass laterally through thehousing 202 such that each male port 230 extends from both the tubingend 204 and the engagement end 206. On the tubing end 204, each maleport 230 comprises a barb 234 for interfacing with a gas line, such asthe gas lines comprising the tubing 30 of FIG. 1. To place the male port230 in communication with a tubing, a corresponding gas line of thetubing is placed over the barb 234. While in this embodiment, the gaslines of the tubing 30 are secured using the barb 234, other forms ofconnection for gas lines may be used, such as TC connections, Luerconnections, and the like.

On the engagement end 206, each male port 230 further comprises astepped section 250 which is configured to engage with a correspondingfemale port 280 in an interface 260 on a pneumatic controller, such asthe controller 40 of FIG. 1. Further, the male ports 230 comprise achannel 232 extending from the barb 234 to the stepped section 250,which may allow for the passage of gas, liquid, or other substancesbetween the tubing 30 and controller 40. At least one of the male ports230 may be designated for a particular function, such as a vacuum port242. Further, a pair of tabs 208 extend laterally from each side of thehousing 202 on the tubing end 204. The tabs 208 may be used, forexample, for gripping the connector 200 to engage or disengage theconnector 200 from the corresponding interface 260, which may beperformed either manually, or by automation with appropriatelyconfigured hardware.

The housing 202 may further comprise a post 210 comprising a connectormagnet 212 positioned on the engagement end 206. In this embodiment, thehousing 202 comprises two posts 210 at each side of the male ports 230,each post 210 comprising a connector magnet 212. However, in certainembodiments, the housing 202 may comprise a single post, multiple posts,lack a post, or include posts without magnets. Similarly, in certainembodiments, the housing 202 may comprise a single magnet, multiplemagnets, lack a magnet, or include magnets without posts. In theembodiment shown, the connector magnets 212 are shaped similarly to theattached surface of the post 210; however, a variety of magnets andshapes may be used.

In the embodiment shown, each post 210 and connector magnet 212 areshaped to be received by a corresponding aperture 264 containing areceiving magnet 266 in the interface 260. An attractive force betweeneach connector magnet 212 and receiving magnet 266 may be used to securethe connector 200 to the interface 260, thus placing the male ports 230in fluid communication with the female ports 280. Further, the magnets212, 266 may be used to help properly align and place the connector 200over the interface 260. For example, the polarity of the connectormagnets 212 may be configured to be the same as the polarity of thereceiving magnets 266 when the connector 200 is positioned over theinterface 260 backwards or in an otherwise incorrect position, thusresulting in a resistive force preventing the connector 200 fromengaging with the interface 260. However, in certain embodiments, eitherthe connector magnets 212 or receiving magnets 266 may simply comprise apiece of metal. In these embodiments, if proper alignment is desired,other forms of engagement may be used, e.g. by keying or spacing theplacement of the male ports 230 and female ports 280 such that theconnector 200 may only engage with the interface 260 in a singleposition. For example, as shown in FIG. 13A, the spacing between thevacuum port 242 and adjacent male port 230 may be slightly wider thanthe spacing between the other male ports 230. Similarly, in certainembodiments, the size and/or shape of the apertures 264 may varycompared to one another to accept only a particular post 210 and/orconnector magnet 212 on a connector 200. Various embodiments andconfigurations are within the scope of the present disclosure.

In certain embodiments, other forms of securing engagement, as opposedto magnetic force, may be used to secure the connector 200 to theinterface 260. For example, the connector 200 may use an existingin-line vacuum force, as in the connector 150 of FIG. 8, topneumatically secure the connector to the interface. Alternately, othermechanical means may be used to secure the connector 200 to theinterface 260, such as screws, thumb screws, bolts, and the like. Forexample, thumb screws may be preferable in some embodiments, as itprovides a reliable connection between the connector 200 and interface260 that is less likely to be accidentally dislodged. However, inembodiments using automation, pneumatics and/or magnetic coupling may bepreferable, as less force is needed to disengage and engage theconnector 200.

Referring to FIGS. 13A-B and FIG. 14, in this embodiment, the housing202 further comprises a plurality of bores 216 passing through thehousing 202, such that the bores 216 are open to the tubing end 204 andthe engagement end 206. Each bore 216 may further comprise a fittingsection 218 for receiving and securing one of the male ports 230 to thebore 216, and an open section 220 proximate the engagement end 206. Incertain embodiments, the fitting section 218 may comprise additionalfeatures for receiving and securing a male port 230, such as threads,grooves, tapering, and the like. In the embodiment shown, the fittingsection 218 has a thinner diameter than the open section 220, and thefitting section 218 and open section 220 are axial with respect to oneanother. Further, a bore 216 may be intended for a particular function,such as vacuum, for the vacuum port 242. This intention may bedesignated on the housing 202 by a structural feature or indicator 214,such as a raised surface on the tubing end 204. Alternately, the housing202 may use other features, such as markings on the tubing end 204 or akeyed spacing or arrangement of ports, to designate the use of bores andports for a particular function. In the embodiment shown, the vacuumport 242 is also colored differently from the male ports 230 as anindication of its intended use.

In the embodiment shown, the plurality of male ports 230 are positionedwithin the bores 216. Each male port 230 may comprise two separatepieces, a syringe 252 and a filter 244, which are configured to engagewith one another to form the male port 230. When engaged together, thechannel 232 (as shown in FIGS. 12A-B) extends through the syringe 252and filter 244. In this embodiment, the syringe 252 and filter 244 eachmay comprise a body having several components. The syringe 252 comprisesthe barb 234, a bolt 236, a threaded segment 238, and a tapered segment240. To position the syringe 252 within one of the bores 216, thetapered segment 240 is placed within the fitting section 218 via thetubing end 204 of the housing 202. The syringe 252 is then rotated bygripping the bolt 236, causing the threaded segment 238 to engage withthe inner surface of the fitting section 218. The syringe 252 isproperly positioned within the bore 216 when the bolt 236 is in contactwith the surface of the tubing end 204, thus securing the syringe 252within the bore 216.

While in this embodiment, the syringe 252 and filter 244 are separable,in certain embodiments, these elements may comprise a single component.Further, while the barb 234, bolt 236, threaded segment 238, and taperedsegment 240 are arranged in this order along the syringe 252, theseelements may be arranged in alternate ways to accommodate alternateembodiments of bores 216 and/or housings 202 according to thedisclosure. For example, in certain embodiments, a bolt 236 may beplaced below a threaded segment 238 so that the syringe 252 may bepositioned within the bore 216 from the engagement end 206. Similarly,in certain embodiments, the threaded segment 238 may comprise otherfeatures, such as grooves or tapering, for securing the syringe 252within the bore 216. In still further embodiments, the various featuresof the syringe 252 and filter 244 may be molded as part of the housing202, for example, such that the channel 232 is an integral component ofthe housing 202. Various embodiments are considered to be within thescope of the disclosure.

As noted above, each male port 230 may comprise two separate pieces, asyringe 252 and a filter 244. The filter 244 may be configured to engagewith the syringe 252, for example, by using a Luer-style connection(such as a Luer slip or Luer lock), threads, or other form ofengagement. In this embodiment, the filter 244 comprises a receivingsection 246, a filter element 248 disposed within the channel 232, andthe stepped section 250. The portion of the channel 232 within thereceiving section 246 may be tapered to receive the tapered segment 240of the syringe 252. Thus, to secure the filter 244 to the syringe 252,the filter 244 is positioned within the open section 220 of the bore 216such that the receiving section 246 of the filter 244 receives thetapered segment 240 of the syringe 252. The filter 244 is then pressedagainst the syringe 252, securing the filter 244 to the syringe 252 byfriction and creating a fluid tight seal.

In this embodiment, the filter 244 comprises the filter element 248disposed within the channel 232. The filter element 248 may comprise anykind of filter, such as hydrophobic filters and PTFE filters. In thisway, the filters 244 may allow passage of air and other gases, butprevent the passage of water and other fluids. The size, shape, and kindof filters 244 may also vary depending on a desired flow rate or otherparameters. For example, in this embodiment, the filters 244 comprisenine 4 mm 0.45 μm PTFE filters and one 13 mm 0.45 μm PTFE filter. Thesingle 13 mm diameter filter may be used for a vacuum line connected tothe vacuum port 242, which may benefit from a higher air flow rate.Filters may comprise, for example, Millex® syringe filters, commerciallyavailable from EMD Millipore Corporation. However, in certainembodiments, a filter 244 may lack a filter element 248, and thus allowpassage of either gas or liquid.

In this embodiment, the filters 244 are replaceable. In someembodiments, filters may be replaced by ejecting each of the filters 244and replacing them with a new set. In certain embodiments, filters maybe replaced by ejection and replacement with a new set, e.g., usingmechanical means. Similarly, in certain embodiments, filters 244 may besimultaneously attached, e.g., by placing the connector 200 onto anarray of filters 244 appropriately spaced to receive each of the taperedportions of the corresponding syringes. However, in still furtherembodiments, filters 244 may be permanently connected to a connector200. Various embodiments and configurations are considered to be withinthe scope of the disclosure.

The connector 200 is configured to engage with a corresponding interface260, which may be located on either side of the tubing 30, such as on amanifold or controller. For example, a controller, such as thecontroller 40 of FIG. 1, may further comprise an interface 260configured to receive the connector 200. In the embodiment shown inFIGS. 13A-B and FIG. 14, the interface 260 comprises a plurality offemale ports 280 which receive the male ports 230 of the connector 200,placing the female ports 280 and male ports 230 in fluid communication.Each female port 280 comprises a seal 282 positioned above an opening284 in communication with a channel 286 of the pneumatic controller 40.Each channel 286 may be configured to supply a liquid, gas, or othersubstance to the female ports 280. In the embodiment shown, each channel286 is configured to supply variable pneumatic pressure from thecontroller 40. Accordingly, when the male ports 230 of the connector 200are in communication with the female ports 280 of the interface 260, thechannel 232 of the male port 230 is in communication with the channel286 of the controller 40. Accordingly, the tubing 30 is in fluidcommunication with the channels 286 of the controller 40 via theconnector 200.

Seals 282 are used to fluidly separate each female port 280, andaccordingly each channel 286, from one another. The seals 282 may beretained by a panel 262. In the embodiment shown, the panel 262comprises openings for each of the female ports 280 and apertures 264.In certain embodiments, the seals 282 may be positioned within groovesdefined within the openings 284 of the female port 280, which may eithercomplement or replace the panel 262. Seals 282 may comprise, forexample, O-rings, which may further comprise a “U”-shaped cross-sectionto allow for low insertion force.

In the embodiment shown in FIGS. 12A and 13A, the connector 200 may beinitially separated and disengaged from the interface 260 on thecontroller 40. As shown in the embodiments of FIGS. 12B and 13B, theconnector 200 is engaged with the interface 260 when the engagement end206 is brought into contact with the interface 260 such that the post210 with the connector magnet 212 enters the corresponding aperture 264with the receiving magnet 266, thus using magnetic attraction to securethe connector 200 to the interface 260. Engaging the connector 200 tothe interface 260 further causes each male port 230 to enter acorresponding female port 280, thus placing each gas line of the tubing30 in fluid communication with the channels 286 of the controller.Further, each seal 282 is placed in contact with the stepped section 250of the corresponding filter 244, substantially preventing fluidcommunication between each channel 286. Thus, the controller 40 cansupply precise levels of variable pressure, including vacuum, to acorresponding manifold downstream to control a microfluidic process orexperiment in a microfluidic plate.

Similar to the pneumatic connector 150, the pneumatic connector 200results in a variety of advantages, such as ease of cleaning,transportation of a manifold and tubing, reducing operator workflow,applicability to automation, and identification of incomplete orimperfect seals. Additionally, the use of a plurality of filters 244 ina single connector 200 has a significant advantage in that all of thefilters 244 may be simultaneously removed from the controller concurrentwith disengaging the connector, as opposed to individually removing eachfilter. Thus, the connector 200 provides a fast, nearly automaticconnection to the controller.

Moreover, the use of filters 244, such as hydrophobic filters, in theremovable connector 200 between the tubing 30 and controller 40 hasadditional advantages. For example, if a liquid backflows from themanifold 120 through the tubing 30, filters 244 prevent the liquid fromentering the channels 286, potentially harming or contaminating thecontroller 40. Filters 244 may also be used to prevent contamination ofthe tubing 30 and a downstream manifold and microfluidic plate, such asthe microfluidic plate 100 and manifold 120 attached to the tubing 30 ofFIGS. 4A-B. Moreover, filters 244 using a slip, threaded, or other formof removable connection may be single-use, helping to preventcontamination each time the connector 200 is secured to the interface260 of the controller 40.

A connector 200 incorporating a plurality of filters 244 may also beused for efficiently cleaning both the manifold 120 and tubing 30.Conventional cleaning methods of the gas lines and tubing associatedwith pneumatic control of microfluidic devices typically involveaspirating a cleaning solution into a syringe, and then injecting thecleaning solution into individual lines. In contrast, the controller 40may be configured to aspirate a cleaning solution, such as hydrogenperoxide, into the tubing 30, thus cleaning all of the gas linescomprising the tubing 30 simultaneously.

Microfluidic Cleaning Plate and Method of Use

FIGS. 15-17 illustrate a cleaning plate 300 for cleaning a manifold andtubing according to an embodiment of the disclosure. The cleaning plate300 may comprise a variety of materials, such as PDMS, molded plastic,and the like. The cleaning plate 300 has similar dimensions to acorresponding microfluidic plate, such as the microfluidic plate 100 ofFIG. 2. Accordingly, in the embodiment shown in FIG. 17, a manifold,such as the manifold 120 of FIGS. 4A-B, may be positioned over thecleaning plate 300 such that it is in fluid communication with thecleaning plate 300, just as the manifold 120 would be positioned overand enter fluid communication with a microfluidic plate 100. Further, acontroller may deliver vacuum to the cleaning plate 300 via a vacuumchannel to seal the manifold to the plate.

In the embodiment shown in FIGS. 15-16, the cleaning plate 300 furthercomprises sidewalls 302 that are raised from a surface 304 of thecleaning plate 300. The sidewalls 302 come into contact with themanifold when the manifold is sealed to the plate. The cleaning plate300 may comprise a plurality of wells, which in the embodiment showncomprise a central well 306, gas line well 308, and vacuum line well310. The central well 306 and gas line well 308 may be filled with acleaning solution, such as a hydrogen peroxide solution, an alcoholsolution, and the like. The cleaning plate 300 further comprises aplurality of cleaning solution channels 312. In the embodiment shown inFIG. 17, which illustrates the manifold 120 of FIG. 5 aligned over thecleaning plate 300 of FIG. 16, the gas line well 308 and vacuum linewell 310 are positioned beneath the outlets for the gas environmentchannel 128 and vacuum channel 130 of the manifold 120, respectively.Similarly, the cleaning solution channels 312 are positioned beneath thechannel outlets 126 of the manifold. Referring to FIGS. 15-16, thecleaning solution channels 312 further comprise openings 314, which arein fluid communication with a plurality of transfer channels 316 influid communication with the central well 306. In this embodiment, thetransfer channels 316 comprise four cavities in the base of the centralwell 306 and an internal channel (not shown) rising up from the base andin communication with the openings 314 within the cleaning solutionchannels 312.

In this embodiment, the sidewalls of the central well 306, gas line well308, and cleaning solution channels 312 rise to the same height as thesidewalls 302 of the cleaning plate 300, and thus are fluidly separatedfrom one another when the manifold 120 is sealed to the plate. Incontrast, the sidewalls of the vacuum line well 310 only rise to thesurface 304 of the plate. Thus, to seal a manifold to the cleaning plate300 (in the embodiment shown in FIG. 17), the manifold is aligned overand placed on top of the plate and pressed down against the plate. Avacuum line in communication with the manifold 120 is then activated.Activating the vacuum line seals the manifold 120 to the plate bycreating a vacuum in the volume between the surface 304, sidewalls 302,and the sidewalls of the central well 306, gas line well 308, andcleaning solution channels 312.

Once the manifold 120 has been sealed to the cleaning plate 300, acleaning sequence may be performed which aspirates cleaning solutionplaced in the wells of the cleaning plate 120 into the manifold and thetubing between the manifold 120 and the controller, such as thecontroller 40 of FIG. 1. First, the central well 306 and gas line well308 may be filled with a cleaning solution. The manifold 120 is thenplaced over the cleaning plate 300, and a cleaning protocol may beactivated on the controller 40. The controller delivers negativepressure to each of the channels within the manifold 120. The negativepressure aspirates cleaning solution from the central well 306 into thetransfer channels 316, through the cleaning solution channels 312, andinto the outlets 126 of the manifold. Similarly, cleaning solution fromthe gas line well 308 is aspirated into the outlet for the gasenvironment channel 128 of the manifold. As the cleaning solutiontraverses the channels of the manifold 120, the cleaning solutioncontinues into the tubing 30, thus cleaning each of the gas linescomprising the tubing 30. Finally, the cleaning solution is stopped bythe filter 244 of the connector 200 (as shown in the embodiment of FIGS.12A-B), thus maximizing cleaning of the length of the tubing 30,manifold 120, and any intermediate components. Further, because thefilter 244 stops the flow of the cleaning solution, the connector 200also minimizes any risk of damage to the controller 40 as a result ofthe cleaning protocol.

Once the cleaning process is complete, it may be reversed such that thecleaning solution is returned back into the cleaning plate 300. Themanifold 120 may then be disconnected from the cleaning plate 300. Themanifold is then ready to use for attachment to a microfluidic plate foran experiment. If the filters 244 of the connector 200 are single-use,they may be replaced.

Further, it should be noted that various features of the aboveembodiments and disclosure may be combined with one another to formvarious pneumatic connectors, pneumatic manifolds, microfluidic plates,cleaning plates, and microfluidic control and analysis systems. Thepresent disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, other various embodiments of andmodifications to the present disclosure, in addition to those describedherein, will be apparent to those of ordinary skill in the art from theforegoing description and accompanying drawings.

Thus, such other embodiments and modifications are intended to fallwithin the scope of the present disclosure. Furthermore, although thepresent disclosure has been described herein in the context of aparticular implementation in a particular environment for a particularpurpose, those of ordinary skill in the art will recognize that itsusefulness is not limited thereto and that the present disclosure may bebeneficially implemented in any number of environments for any number ofpurposes. Accordingly, the claims set forth below should be construed inview of the full breadth and spirit of the present disclosure asdescribed herein.

What is claimed is:
 1. A removable pneumatic connector, comprising: abody having a plurality of bores passing through, each bore surroundedby a sealing member on an inner surface of the body; a vacuum portdisposed on the inner surface of the body; and an outer seal on theinner surface of the body surrounding the sealing members and the vacuumport; wherein the outer seal and each of the sealing members areconfigured to create a vacuum holding area in the volume between theouter seal and each of the holding members when the inner surface of thebody is placed against a substrate.
 2. The removable pneumatic connectorof claim 1, further comprising a plurality of gas lines, wherein eachline of the plurality of gas lines is placed within a correspondingbore.
 3. The removable pneumatic connector of claim 2, furthercomprising a vacuum line placed within the vacuum port.
 4. The removablepneumatic connector of claim 3, wherein the vacuum line is configured todeliver negative pressure to the vacuum port, such that a vacuum iscreated within the vacuum holding area.
 5. The removable pneumaticconnector of claim 1, wherein the sealing member surrounding each boreand the outer seal have a similar compressibility ratio.
 6. Theremovable pneumatic connector of claim 1, wherein the sealing membersurrounding each bore comprises an O-ring, and the outer seal comprisesa gasket.